Ilona I. Concha

Resolution of the Facilitated Transport of Dehydroascorbic Acid from Its Intracellular Accumulation as Ascorbic Acid

We performed a detailed kinetic analysis of the uptake of dehydroascorbic acid by HL-60 cells under experimental conditions that enabled the differentiation of dehydroascorbic acid transport from the intracellular reduction/accumulation of ascorbic acid. Immunoblotting and immunolocalization experiments identified GLUT1 as the main glucose transporter expressed in the HL-60 cells. Kinetic analysis allowed the identification of a single functional activity involved in the transport of dehydroascorbic acid in the HL-60 cells. Transport was inhibited in a competitive manner by both 3-O-methyl-D-glucose and 2-deoxy-D-glucose. In turn, dehydroascorbic acid competitively inhibited the transport of both sugars. A second functional component identified in experiments measuring the accumulation of ascorbic acid appears to be associated with the intracellular reduction of dehydroascorbic acid to ascorbic acid and is not directly involved in the transport of dehydroascorbic acid via GLUT1. Transport of dehydroascorbic acid by HL-60 cells was independent of the presence of external Na, whereas the intracellular accumulation of ascorbic acid was found to be a Na-sensitive process. Thus, the transport of dehydroascorbic acid via glucose transporters is a Na-independent process which is kinetically and biologically separable from the reduction of dehydroascorbic acid to ascorbic acid and its subsequent intracellular accumulation.

Andrographolide interferes with binding of nuclear factor-κB to DNA in HL-60-derived neutrophilic cells

1 Andrographolide, the major active component from Andrographis paniculata, has shown to possess anti‐inflammatory activity. Andrographolide inhibits the expression of several proinflammatory proteins that exhibit a nuclear factor kappa B (NF‐κB) binding site in their gene. 2 In the present study, we analyzed the effect of andrographolide on the activation of NF‐κB induced by platelet‐activating factor (PAF) and N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) in HL‐60 cells differentiated to neutrophils. 3 PAF (100 nM) and fMLP (100 nM) induced activation of NF‐κB as determined by degradation of inhibitory factor B α (IκBα) using Western blotting in cytosolic extracts and by binding to DNA using electrophoretic mobility shift assay (EMSA) in nuclear extracts. 4 Andrographolide (5 and 50 μM) inhibited the NF‐κB‐luciferase activity induced by PAF. However, andrographolide did not reduce phosphorylation of p38 MAPK or ERK1/2 and did not change IκBα degradation induced by PAF and fMLP. 5 Andrographolide reduced the DNA binding of NF‐κB in whole cells and in nuclear extracts induced by PAF and fMLP. 6 Andrographolide reduced cyclooxygenase‐2 (COX‐2) expression induced by PAF and fMLP in HL‐60/neutrophils. 7 It is concluded that andrographolide exerts its anti‐inflammatory effects by inhibiting NF‐κB binding to DNA, and thus reducing the expression of proinflammatory proteins, such as COX‐2.

PRESENCE OF RNA IN THE SPERM NUCLEUS

The RNase-colloidal gold procedure for the ultrastructural localization of RNA was used for rat testis. Along with other structures, it was found that the testicular sperm nucleus was well stained. Similar labelling was observed in the nucleus of rat epididymal sperm and human sperm. The RNA was extracted from sperm and analyzed by electrophoresis on 10% polyacrylamide gel and 7 M urea. The electrophoretic profile revealed a complex set of bands ranging in size from tRNA to high molecular weight components. On the average, a content of about 0.1 pg of RNA per rat or human sperm was found.

AβPP induces cdk5-dependent tau hyperphosphorylation in transgenic mice Tg2576

Previous studies of Abeta-induced neuronal damage of hippocampal cells in culture have provided strong evidence that deregulation of the Cdk5/p35 kinase system is involved in the neurodegeneration pathway. Cdk5 inhibitors and antisense probes neuroprotected hippocampal cells against the neurotoxic action of Abeta. To further investigate the mechanisms underlying the participation of Cdk5 in neuronal degeneration, the transgenic mouse containing the Swedish mutations, Tg2576, was used as an animal model. Immunocytochemical studies using anti-Abeta(1-17) antibody evidenced the presence of labeled small-clustered core plaques in the hippocampus and cortex of 18-month-old transgenic mice brains. The loss of granular cells without a compressed appearance was detected in the vicinity of the cores in the dentate gyrus of the hippocampus. Immunostaining of Tg2576 brain sections with antibodies AT8, PHF1 and GFAP labeled punctuate dystrophic neurites in and around the amyloid core. Reactive astrogliosis around the plaques in the hippocampus was also observed. Studies at the molecular level showed differences in the expression of the truncated Cdk5 activator p25 in the transgenic animal, as compared with wild type controls. However no differences in Cdk5 levels were detected, thus corroborating previous cellular findings. Interestingly, hyperphosphorylated tau epitopes were substantially increased as assessed with the AT8 and PHF1 antibodies, in agreement with the observation of a p25 increase in the transgenic animal. These observations strongly suggest that the increased exposure of Alzheimers type tau phosphoepitopes in the transgenic mice correlated with deregulation of Cdk5 leading to an increase in p25 levels. These studies also provide further evidence on the links between extraneuronal amyloid deposition and tau pathology.

Hexose transporter expression and function in mammalian spermatozoa: Cellular localization and transport of hexoses and vitamin C

We analyzed the expression of hexose transporters in human testis and in human, rat, and bull spermatozoa and studied the uptake of hexoses and vitamin C in bull spermatozoa. Immunocytochemical and reverse transcription‐polymerase chain reaction analyses demonstrated that adult human testis expressed the hexose transporters GLUT1, GLUT2, GLUT3, GLUT4, and GLUT5. Immunoblotting experiments demonstrated the presence of proteins of about 50–70 kD reactive with anti‐GLUT1, GLUT2, GLUT3, and GLUT5 in membranes prepared from human spermatozoa, but no proteins reactive with GLUT4 antibodies were detected. Immunolocalization experiments confirmed the presence of GLUT1, GLUT2, GLUT3, GLUT5, and low levels of GLUT4 in human, rat, and bull spermatozoa. Each transporter isoform showed a typical subcellular localization in the head and the sperm tail. In the tail, GLUT3 and GLUT5 were present at the level of the middle piece in the three species examined, GLUT1 was present in the principal piece, and the localization of GLUT2 differed according of the species examined. Bull spermatozoa transported deoxyglucose, fructose, and the oxidized form of vitamin C, dehydroascorbic acid. Transport of deoxyglucose and dehydroascorbic acid was inhibited by cytochalasin B, indicating the direct participation of facilitative hexose transporters in the transport of both substrates by bull spermatozoa. Transport of fructose was not affected by cytochalasin B, which is consistent for an important role for GLUT5 in the transport of fructose in these cells. The data show that human, rat, and bull spermatozoa express several hexose transporter isoforms that allow for the efficient uptake of glucose, fructose, and dehydroascorbic acid by these cells. J. Cell. Biochem. 71:189–203, 1998.

A metabolic switch in brain: glucose and lactate metabolism modulation by ascorbic acid

In this review, we discuss a novel function of ascorbic acid in brain energetics. It has been proposed that during glutamatergic synaptic activity neurons preferably consume lactate released from glia. The key to this energetic coupling is the metabolic activation that occurs in astrocytes by glutamate and an increase in extracellular [K+]. Neurons are cells well equipped to consume glucose because they express glucose transporters and glycolytic and tricarboxylic acid cycle enzymes. Moreover, neuronal cells express monocarboxylate transporters and lactate dehydrogenase isoenzyme 1, which is inhibited by pyruvate. As glycolysis produces an increase in pyruvate concentration and a decrease in NAD+/NADH, lactate and glucose consumption are not viable at the same time. In this context, we discuss ascorbic acid participation as a metabolic switch modulating neuronal metabolism between rest and activation periods. Ascorbic acid is highly concentrated in CNS. Glutamate stimulates ascorbic acid release from astrocytes. Ascorbic acid entry into neurons and within the cell can inhibit glucose consumption and stimulate lactate transport. For this switch to occur, an ascorbic acid flow is necessary between astrocytes and neurons, which is driven by neural activity and is part of vitamin C recycling. Here, we review the role of glucose and lactate as metabolic substrates and the modulation of neuronal metabolism by ascorbic acid.

Andrographolide reduces IL-2 production in T-cells by interfering with NFAT and MAPK activation

The nuclear factor of activated T cells (NFAT) is a transcription factor essential for cytokine production during T-cell activation and is the target of several immunosuppressive drugs. Andrographolide is a diterpenic labdane that possesses anti-inflammatory and immunomodulatory effects. Several studies propose that andrographolide can reduce the immune response through inhibition of the nuclear factor kappa B (NF-kappaB) and mitogen-activated protein kinases (MAPK) such as extracellular signal regulated kinase 1/2 (ERK1/2) pathways. Moreover, andrographolide reduces IFN-gamma and IL-2 production induced by concanavalin A in murine T-cell. Nevertheless, the mechanisms involved in the decrease of cytokine production are unknown. In the present study, we determined that andrographolide reduced IL-2 production in Jurkat cells stimulated with phorbol myristate acetate and ionomycin (PMA/Ionomycin). We then showed that andrographolide reduced NFAT luciferase activity and interfered with its nuclear distribution, with these effects being linked to an increase in c-jun-N-terminal kinase (JNK) phosphorylation. Additionally, reduction of NF-kappaB activity in Jurkat cells treated with andrographolide was observed. Using Western blotting, we demonstrated that andrographolide decreased ERK1 and ERK5 phosphorylation induced by anti-CD3 or PMA/Ionomycin. Andrographolide did not affect cell viability at concentration of 10 and 50 muM; however, our results suggest that andrographolide increase early apoptosis at 100 muM. We concluded that andrographolide can exert immunomodulatory effects by interfering with NFAT activation and ERK1 and ERK5 phosphorylation in T-cells.

Gluconeogenesis-Linked Glycogen Metabolism Is Important in the Achievement of In Vitro Capacitation of Dog Spermatozoa in a Medium Without Glucose

Abstract In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metabolic substrate (l-CCM) was accompanied by a progressive increase of intracellular glycogen during the first 2 h of incubation, which was followed by a subsequent decrease of glycogen levels after up to 4 h of incubation. Lactate from the medium is the source for the observed glycogen synthesis, as the presence of [14C]glycogen after the addition to l-CCM with [14C]lactate was demonstrated. The existence of functional gluconeogenesis in dog sperm was also sustained by the presence of key enzymes of this metabolic pathway, such as fructose 1,6-bisphophatase and aldolase B. On the other hand, glycogen metabolism from gluconeogenic sources was important in the maintenance of a correct in vitro fertilization after incubation in the l-CCM. This was demonstrated after the addition of phenylacetic acid (PAA) to l-CCM. In the presence of PAA, in vitro capacitation of dog spermatozoa suffered alterations, which translated into changes in capacitation functional markers, like the increase in the percentage of altered acrosomes, a distinct motion pattern, decrease or even disappearance of capacitation-induced tyrosine phosphorylation, and increased heterogeneity of the chlorotetracycline pattern in capacitated cells. Thus, this is the first report indicating the existence of a functional glyconeogenesis in mammalian spermatozoa. Moreover, gluconeogenesis-linked glycogen metabolism seems to be of importance in the maintenance of a correct in vitro capacitation in dog sperm in the absence of hexoses in the medium.

Ascorbic acid participates in a general mechanism for concerted glucose transport inhibition and lactate transport stimulation

In this paper, we present a novel function for ascorbic acid. Ascorbic acid is an important water-soluble antioxidant and cofactor in various enzyme systems. We have previously demonstrated that an increase in neuronal intracellular ascorbic acid is able to inhibit glucose transport in cortical and hippocampal neurons. Because of the presence of sodium-dependent vitamin C transporters, ascorbic acid is highly concentrated in brain, testis, lung, and adrenal glands. In this work, we explored how ascorbic acid affects glucose and lactate uptake in neuronal and non-neuronal cells. Using immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, the expression of glucose and ascorbic acid transporters in non-neuronal cells was studied. Like neurons, HEK293 cells expressed GLUT1, GLUT3, and SVCT2. With radioisotope-based methods, only intracellular ascorbic acid, but not extracellular, inhibits 2-deoxyglucose transport in HEK293 cells. As monocarboxylates such as pyruvate and lactate, are important metabolic sources, we analyzed the ascorbic acid effect on lactate transport in cultured neurons and HEK293 cells. Intracellular ascorbic acid was able to stimulate lactate transport in both cell types. Extracellular ascorbic acid did not affect this transport. Our data show that ascorbic acid inhibits glucose transport and stimulates lactate transport in neuronal and non-neuronal cells. Mammalian cells frequently present functional glucose and monocarboxylate transporters, and we describe here a general effect in which ascorbic acid functions like a glucose/monocarboxylate uptake switch in tissues expressing ascorbic acid transporters.

Different involvement for aldolase isoenzymes in kidney glucose metabolism: Aldolase B but not aldolase A colocalizes and forms a complex with FBPase

The expression of aldolase A and B isoenzyme transcripts was confirmed by RT‐PCR in rat kidney and their cell distribution was compared with characteristic enzymes of the gluconeogenic and glycolytic metabolic pathway: fructose‐1,6‐bisphosphatase (FBPase), phosphoenol pyruvate carboxykinase (PEPCK), and pyruvate kinase (PK). We detected aldolase A isoenzyme in the thin limb and collecting ducts of the medulla and in the distal tubules and glomerula of the cortex. The same pattern of distribution was found for PK, but not for aldolase B, PEPCK, and FBPase. In addition, co‐localization studies confirmed that aldolase B, FBPase, and PEPCK are expressed in the same proximal cells. This segregated cell distribution of aldolase A and B with key glycolytic and gluconeogenic enzymes, respectively, suggests that these aldolase isoenzymes participate in different metabolic pathways. In order to test if FBPase interacts with aldolase B, FBPase was immobilized on agarose and subjected to binding experiments. The results show that only aldolase B is specifically bound to FBPase and that this interaction was specifically disrupted by 60 μM Fru‐1,6‐P2. These data indicate the presence of a modulated enzyme–enzyme interaction between FBPase and isoenzyme B. They affirm that in kidney, aldolase B specifically participates, along the gluconeogenic pathway and aldolase A in glycolysis.